Endocannabinoids (ECs) represent a unique group of lipids that function as chemical messengers in the nervous system. To date, the two principle ECs identified in mammals are N-arachidonoyl-ethanolamine (anandamide) and 2-arachidonoyl-glycerol (2-AG). They have been implicated in various physiological and pathological functions including appetite, pain, sensation, memory, and addiction (1). Unlike traditional neurotransmitters, which are stored in vesicles, ECs are synthesized and released on demand, and then rapidly degraded to terminate signaling. Thus, the metabolic pathways that govern EC turnover are critical in determining the magnitude and duration of neuronal signaling events (2). Endocannabinoid biosynthesis, in contrast to degradation, is poorly understood. Recently, two serine hydrolases, DAGL-a and DAGL-B, were cloned and found to selectively cleave sn-1 acyl chains from diacylglycerols (DAG) to generate 2-AG in vitro (3). Their function in the nervous system was validated in vivo by the generation of DAGL-a and DAGL-B knock-out mice (4, 5). However, it is still unclear to what extent DAGL-a/B catalytic activity contributes to 2-AG-mediated signaling. The development of potent and selective inhibitors would offer a means to perturb DAGL-a/B activity in a selective, reversible, and temporally-controlled manner. Given the non-selective nature of current DAGL-a/B inhibitors (6), specific chemical probes would serve as invaluable tools to delineate DAGL-a/B function in 2-AG signaling networks of the brain.

Assay Overview:The purpose of this assay is to determine whether test compounds can inhibit antitarget DAGLa in a gel-based competitive activity-based proteomic profiling (ABPP) assay. In this assay, the antitarget overexpressed DAGLa is incubated with test compound followed by reaction with a fluorescently-labeled probe HT-01, which labels a handful of serine hydrolases, including DAGLa. The reaction products are separated by SDS-PAGE and visualized in-gel using a flatbed fluorescence scanner. The percentage activity remaining is determined by measuring the integrated optical density (IOD) of the bands. As designed, test compounds that act as DAGLa inhibitors will prevent enzyme-probe interactions, thereby decreasing the proportion of bound fluorescent probe, giving lower fluorescence intensity in the band in the gel.Protocol Summary:Membrane proteome of transiently transfected 293T Hek cells overexpressing mouse DAGLa (50 uL of 2 mg/mL) in Dulbecco's PBS (DPBS) was treated with test compound (1 uL of a 50x stock in DMSO; 10 uM, 2 uM, 0.4 uM, 0.08 uM, or 0.016 uM final concentration) or DMSO (1 uL) for 30 minutes at 37 C. The activity-based probe HT-01 (1 uL of a 50x stock in DMSO; 1 uM final concentration) was added, and the reaction was incubated for 30 minutes at 37 C, quenched with an equal volume of 2x SDS-PAGE loading buffer (reducing), separated by SDS-PAGE, and visualized by in-gel fluorescent scanning. The percentage of DAGLa activity remaining was determined by measuring the integrated optical density of the individual protein bands relative to the DMSO-only (no compound) control.The % inhibition was then calculated as follows:%_Inhibition = ( 1 -( IOD_Test_Compound -Median_IOD_Low_Control ) / ( Median_IOD_High_Control - Median_IOD_Low_Control ) ) * 100Where:Test_Compound is defined as DAGLa treated with test compound.High_Control is defined as DAGLa treated with DMSO only (no compound).Low_Control is defined as background in a blank region of the gel.PubChem Activity Outcome and Score:Compounds with less than or equal to 50% inhibition at 0.4 uM compound concentration were considered active. Compounds with less than 50% inhibition at 0.4 uM compound concentration were considered inactive.The reported PubChem Activity Score has been normalized to 100% observed inhibition at 0.4 uM. Negative % inhibition values are reported as activity score zero.The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 80-0.List of Reagents:293T Hek cells overexpressing mouse DAGLa (Open Biosystems Accession BC148308; provided by Assay Provider)FP-Rh (provided by Assay Provider)DPBS (Cellgro 20-031-CV)